Field of the Invention
The invention relates to optically pumped solid-state pulse lasers which are used in mass spectrometers for ionization by laser desorption, particularly matrix-assisted laser desorption (MALDI). It particularly relates to the blanking out of individual laser pulses or groups of laser pulses, giving special consideration to the requirements of pulsed ionization processes such as the MALDI process.
The invention provides a low-cost, non-complex method whereby individual light pulses or groups of light pulses can be blanked out without subsequent light pulses having a higher energy density as a result of the previous blanking out.
Description of the Related Art
An important type of ionization for biomolecules is ionization by matrix-assisted laser desorption (MALDI), which was developed by M. Karas and K. Hillenkamp about twenty years ago. MALDI ablates and ionizes the analyte molecules, preferably biomolecules, which are contained in highly diluted form in a mixture with molecules of a matrix substance in samples on sample supports, by bombarding them with pulses of laser light, usually UV laser light. Nitrogen lasers were previously the main type of laser for this task. Nowadays, however, solid-state lasers are used because they have a far longer lifetime and higher pulse frequencies. The lasers usually used have neodymium-doped crystals and a tripling of the photon energy by non-linear crystals (e.g. with a target wavelength of 355 nanometers).
Nowadays, the ions which are created in the plasma of each such pulse of laser light are primarily accelerated in specially designed MALDI time-of-flight mass spectrometers (MALDI-TOF-MS) with between 20 and 30 kilovolts and axially injected into a flight path. After passing through the flight path, they encounter an ion measuring system, which measures the mass-dependent arrival time of the ions and their quantity, and then records the digitized measurements in the form of a time-of-flight spectrum. In the past, repetition frequencies of the laser light pulses of between 20 and 60 hertz were used for nitrogen lasers. Solid-state lasers have been used with repetition rates of up to 2,000 light pulses per second. Recently, the applicant for this disclosure developed a MALDI-TOF mass spectrometer with light pulse and spectral acquisition frequencies of 10 kilohertz.
In order to avoid saturation effects of the ion detection, care has to be taken that each pulse of laser light does not produce too many ions, for example only a few thousand per pulse at most. A few hundred to a few thousand individual spectra are therefore summed for a time-of-flight spectrum. The mass spectra can achieve mass resolutions of R=m/Δm=80,000 and more nowadays, where Δm is the width of the ion peak at half height.
This mass resolution is achieved only if the energy density of the laser light pulses is correctly adjusted and as constant as possible from shot to shot, however. The properties of the plasma produced by a laser pulse have a strongly nonlinear dependence on the energy density in the pulse of laser light; the optimum setting of the MALDI conditions for full utilization of the measurement range therefore requires that the energy density of the laser light pulses has unusually high constancy. According to the literature, the ion yield is roughly proportional to the sixth or even seventh power of the energy, see, for instance, the review article “The Desorption Process in MALDI” by Klaus Dreisewerd (Chem. Rev. 2003, 103, 395-425). Changing the energy of the laser light pulse by only one percent is enough to change the ion yield by some six to seven percent; something similar applies for other plasma parameters also.
The energy also determines the pressure in the plasma cloud that is produced in every laser shot, for example; and the pressure-dependent expansion of this plasma determines the initial velocity distribution of the ions. As those skilled in the art of MALDI know, this velocity distribution must be accurately focused in time by selecting a suitable delay before the acceleration into the flight path (delayed extraction) and a suitable profile of the accelerating voltage. Even a slight change in the energy becomes noticeable through a measurable deterioration of the mass resolution.
If the series of laser light pulses has to be interrupted at high pulse sequence frequencies, for example because a target to be aimed at on a sample support has to be moved and spatially realigned, this can lead to a problem with the constancy of the energy density. If the outpulsing, and thus the reduction of the stored inversion, is simply interrupted in time, the continued pumping of the laser crystals can lead to an increase in the population of the upper energy state and thus to a strong increase in the energy in the next pulse of laser light. An extremely harmful multiplication of the energy density of the next laser pulse can result easily here. In principle, it is possible to control the pumping process, but this proves to be fundamentally disadvantageous for the service life and efficiency of the pump diode for high pulse frequencies of 10 kilohertz, for example.
FIG. 1 schematically illustrates the underlying problem. A series of uniform pulses of laser light (a) to (k) is interrupted at position (f) (broken line). Pulse (G), which follows the omitted light pulse (f), has increased energy compared to the other pulses of the series, before the value levels off again to roughly the previous value as from pulse (h) on.
Another possible way to avoid energy increase relates to the temporally fast blanking out of laser pulses by mechanical arrangements and/or electro-optical methods (so-called “pulse pickers”) after the laser beam is generated. Mechanical arrangements are not fast enough, however, especially for the kHz regime of operation; electro-optical methods, on the other hand, require a lot of space and are very costly, comparable with the cost of the laser system itself, which makes this kind of solution fundamentally uneconomical.
The acquisition technique of pulsed ion sources such as MALDI ion sources requires that one or more laser pulses be sometimes omitted, however. For example, when changing from one individual sample to the next on a sample support that holds separate samples, it is necessary to omit several laser pulses in order to avoid obtaining mass spectra of the bare sample support or even damaging the sample support. In imaging mass spectrometry which requires surfaces of thin tissue sections to be scanned, laser pulses must be omitted when one raster line of the image has been scanned and it is necessary to start again from the beginning with the next raster line. The subsequent laser pulse in each case should not show an increase in energy significantly greater than one percent, however. Those of skill in the art will acknowledge that this requirement distinguishes MALDI from other laser applications.
In view of the foregoing, there is a need to provide a method which allows the clocked sequence of laser pulses onto a sample support for ionization by laser desorption to be interrupted for any chosen number of pulses without the first laser pulse onto the sample after the interruption having a disadvantageously increased energy density. Furthermore, there is a need to provide a device which makes such an operating mode easy and inexpensive to achieve.